1. Field of the Invention
The present invention relates to a human CC chemokine protein (i.e., a cytokine having the first two of its four cysteine residues adjacent as indicated by xe2x80x9cCCxe2x80x9d) and to polynucleotides encoding this protein.
2. Background Information
The discovery of IL-8, in 1987, revealed the existence of a novel class of small cytokines, now called chemokines, that are widely studied because of their ability to activate leukocytes and their potential role as mediators of inflammations. A number of different human chemokines have been identified after IL-8, by cloning or biochemical purification and amino acid sequencing. All have four conserved cysteines that form characteristic disulfide bonds, a short amino-terminal and a longer carboxy-terminal sequence. Two subfamilies are distinguished by the arrangement of the first two cysteines, which are either separated by one amino acid (CXC chemokines) or are adjacent (CC chemokines.). Chemokine cDNAs typically encode proteins of 92-99 amino acids in length that are secreted after cleavage of a leader sequence of 20-25 amino acids. Modeling on the basis of the NMR-derived structure of IL-8 suggests that CXC and CC chemokines are folded in a similar manner.
The first human CC chemokine was identified by differential hybridization cloning and was termed LD78 (Obaru, K. Fukuda, M., Maeda, S. and Shimada, K. (1986) J. Biochem. (Tokyo) 99, 885-894.) Several cDNA isoforms of a closely related human chemokine, Act-2, were later described (Miller, M. D. and Krangel, M. S. (1992) Crit. Rev. Immunol. 12, 17-46), and two similar proteins, macrophage inflammatory protein 1xcex1 (MIP-1xcex1) and MIP-1xcex2, were purified form the culture medium of lipopolysaccharide (LPS)-stimulated mouse macrophages (Wolpe, S. D., Davatelis, G. Sherry, B. et al. (1988) J. Exp. Med. 167, 570-581). On the basis of more than 70% amino acid identity, the murine and human proteins are considered as homologs, and the terms human MIP-1xcex1 and MIP-1xcex2 are commonly used instead of LD78 and Act-2. The best characterized CC chemokine is monocyte chemotactic protein 1 (MCP-1), which was purified and cloned from different sources (Miller, M. D. and Krangel, M. S. (1992) Crit. Rev. Immunol. 12, 17-46; Yoshimure, T. Robinson, E. A. Tanaka, S. Appella, E. and Leonardo, E. J. (1989) J. Immunol. 142, 1956-1962; Matsushima, K., Larsen, C. G., DuBois, G. C. and Oppenheim, J. J. (1989) J. Exp. Med. 169, 1485-1490). Other CC chemokines, I-309 (Miller, M. D., Hata, S., De Waal Malafyt, R. and Krangel, M. S. (1989) J. Immunol. 143, 2907-2916), RANTES (Schall, T. J. Jongstra, J., Dyer, B. J. et al. (1988) J. Immunol. 141, 1018-1025) and HC14 (Chang, H. C., Hsu, F., Freeman, G. J., Griffin, J. D. and Reinherz, E. L. (1989) Int. Immunol. 1, 388-397), were purified or cloned as products of activated T cells. HC14, termed MCP-2, was also isolated from osteosarcoma cell cultures (Van Damme, J. Proost, P., Lenaerts, J-P. and Opdenakker, G. (1992) J. Exp. Med. 176, 59-65), along with a novel CC chemokine, MCP-3, which was subsequently cloned and expressed (Minty, A. Chalon, P. Guillemot, J. C. et al. (1993) Eur. Cytokine Netw. 4, 99-110; Opdenakker, G. Froyen, G. Fiten, P., Proost, P. and Van Damme, J.(1993) Biochem. Biophys. Res. Commun. 1991, 535-542). These CC chemokines share a sequence identify with MCP-1 of between 29 and 71% (MCP-2 and MCP-3 have 62-71% identity with MCP-1).
MCP-1, the prototype of the CC chemokine sub-family, is chemotatic for monocytes but not for neutrophils (Yoshimure, T. Robinson, E. A. Tanaka, S. Appella, E. and Leonardo, E. J. (1989) J. Immunol. 142,1956-1962; Matsushima, K., Larsen, C. G., DuBois, G. C. and Oppenheim, J. J. (1989) J. Exp. Med. 169, 1485-1490) and was initially considered to be a counterpart of IL-8. Indeed, monocytes respond to all CC chemokines, as judged from stimulus-dependent [Ca2+]i changes (Miller, M. D. and Krangel, M. S. (1992) Crit. Rev. Immunol. 12, 17-46; Bioschoff, S. C., Krieger, M. Brunner, T. et al. (1993) Eur. J. Immunol. 23, 761-767; McColl, S. R., Hachicha, M., Levasseur, S., Noete, K. and Schall, T. J. (1993) J. Immunol. 150, 4550-4560). MCP-1, MCP-2 and MCP-3 induce monocyte infiltration on intradermal injection into rats and rabbits (Van Damme, J. Proost, P., Lenaerts, J-P. and Opdenakker, G. (1992) J. Exp. Med. 176, 59-65; Zacha, C. O. C., Anderson, A. O., Thompson, H. L. et al. (1990) J. Exp. Med. 171,2177-2182), and MCP-1 also elicits in monocytes a respiratory burst (Miller, M. D. and Krangel, M. S. (1992) Crit. Rev. Immunol. 12, 17-46) and the expression of xcex22 integrins (Jiang, Y., Beller, D. I., Frendl, G. and Graves, D. T. (1992) J. Immunol. 148, 2423-2428).
While the view that CXC chemokines act on neutrophils and CC chemokines act on monocytes apparently remains valid, recent studies have revealed that CC chemokines have a much wider range of biological activities since they can also activate some lymphocytes and, in particular, basophil and eosinophil leukocytes. Thus, there is a continuing need in the art for isolating novel CC chemokines.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a human chemokine xcex2-15 (CKxcex2-15) polypeptide having the amino acid sequence in FIGS. 1A-1C [SEQ ID NO:2] or the amino acid sequence encoded by the cDNA clone deposited as ATCC Deposit Number 97519 on Apr. 25, 1996. The nucleotide sequence determined by sequencing the deposited CKxcex2-15 cDNA clone, which is shown in FIGS. 1A-1C [SEQ ID NO:1], contains an open reading frame encoding a polypeptide of about 149 amino acid residues including an initiation codon at positions 1-3, a leader sequence of about 20 amino acid residues and a deduced molecular weight of about 16 kDa. The 129 amino acid sequence of the predicted mature CKxcex2-15 protein is shown in FIGS. 1A-1C (amino acid residues from about 21 to about 149) and in SEQ ID NO:2 (amino acid residues from about 1 to about 129).
Thus, one aspect of the invention provides isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the chemokine xcex2-15 polypeptide having the complete amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequence encoding the chemokine xcex2-15 polypeptide having the complete amino acid sequence in SEQ ID NO:2 but lacking the N-terminal methionine residue; (c) a nucleotide sequence encoding the mature chemokine xcex2-15 polypeptide having the amino acid sequence at positions from about 1 to about 129 in SEQ ID NO:2; (d) a nucleotide sequence encoding the chemokine xcex2-15 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97519; (e) a nucleotide sequence encoding the mature chemokine xcex2-15 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97519; and (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), or (e) above. Preferably, the nucleic acid molecule will encode the mature polypeptide in SEQ ID NO:2 or encoded by the above-described deposited cDNA.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d), (e), or (f) above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to a nucleotide sequence in (a), (b), (c), (d), (e), or (f), above. The polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a chemokine xcex2-15 polypeptide having an amino acid sequence in (a), (b), (c), (d), or (e), above.
The present invention also relates to recombinant vectors which include the isolated nucleic acid molecules of the present invention and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of CKxcex2-15 polypeptides or peptides by recombinant techniques.
The invention further provides an isolated chemokine xcex2-15 polypeptide having amino acid sequence selected from the group consisting of: (a) a polypeptide comprising amino acids from about xe2x88x9220 to about 129 in SEQ ID NO:2; (b) a polypeptide comprising amino acids from about xe2x88x9219 to about 129 in SEQ ID NO:2 ; (c) a polypeptide comprising amino acids from about 1 to about 129 in SEQ ID NO:2; (d) the amino acid sequence of the chemokine xcex2-15 polypeptide having the complete amino acid sequence including the leader encoded by the cDNA clone contained in ATCC Deposit No. 97519; and (e) the amino acid sequence of the mature chemokine xcex2-15 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97519. The polypeptides of the present invention also include polypeptides having an amino acid sequence with at least 90% similarity, more preferably at least 95% similarity to those described in (a), (b), (c), (d), or (e) above, as well as polypeptides having an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99% identical to those above.
An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a chemokine xcex2-15 polypeptide having an amino acid sequence described in (a), (b), (c), (d) or (e), above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a chemokine xcex2-15 polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention. In another embodiment the invention provides an isolated antibody that binds specifically to a chemokine xcex2-15 polypeptide having an amino acid sequence described in (a), (b), (c), (d) or (e) above.
The present inventors have discovered that CKxcex2-15 is expressed only in tissue of the thymus. FIG. 3. For a number of thymus disorders, significantly higher or lower levels of CKxcex2-15 gene expression can be detected in thymus tissue or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a xe2x80x9cstandardxe2x80x9d CKxcex2-15 gene expression level, i.e., the CKxcex2-15 expression level in thymus tissue or bodily fluids from an individual not having the thymus disorder. Thus, the invention provides a diagnostic method useful during diagnosis of a thymus disorder, which involves (a) assaying chemokine xcex2-15 gene expression level in cells or body fluid of that individual; (b) comparing that chemokine xcex2-15 gene expression level with a standard chemokine xcex2-15 gene expression level, whereby an increase or decrease in the assayed chemokine xcex2-15 gene expression level compared to the standard expression level is indicative of a thymus disorder. An additional aspect of the invention is related to a method for treatment of an individual in need of an increased level of chemokine xcex2-15 activity in the body comprising administering to such an individual a composition comprising an isolated chemokine xcex2-15 polypeptide of the invention.